The dsRNA-dependent eIF-2.alpha. kinase (hereinafter "PKR"), also known as DAI, p68, dsI and dsRNA-PK (Clemens, et al., 1993, J. Interferon Res. 13: 241), is an interferon (IFN)-induced enzyme that mediates, in part, the antiviral and antiproliferative effects of IFN (Pestka, et al., 1987, Ann. Rev. Biochem. 56: 727-777; Hovanessian, et al., 1989, J. Interferon Res. 9: 641-647). Other studies have indicated that PKR may also be involved in the regulation of cell growth and differentiation of some cells, function as a tumor suppressor (Meurs, et al., 1993, Proc. Nat. Acad. Sci. 90: 232-236; Petryshyn, et al., 1994, J. Biol. Chem. 259: 14736-14742; Petryshyn, et al., 1988, Proc. Natl. Acad. Sci. 85: 1427-1431; Judware, et al., 1991, Mol. Cell. Biol. 11: 3259-3267; Koromilas, et al., 1992, Science 257: 1685-1689; Chong, et al., 1992, EMBO J. 11: 1553-1562) and modulate signal transduction (Kumar, et al., 1994, Proc. Natl. Acad. Sci. 91: 6288-6292; Maran, et al., 1994, Science 265: 789-792).
Still other studies have indicated that PKR may be involved in regulating programed cell death (apoptosis) (Young et al., 1996 Proc. Nat. Acad. Sci. 93:12451-12455). The mechanism by which PKR controls protein synthesis in vitro is reasonably understood (Lebleu, et al., 1976, Proc. Natl. Acad. Sci. 73: 3107-3111; Farrell, et al., 1977, Cell 11: 187-200; Levin, et al., 1978, Proc. Natl. Acad. Sci. 75: 1121-1125; Samuel, 1979, Proc. Natl. Acad. Sci. 76: 600-604; Petryshyn, et al., 1983, Methods Enzymol. 99: 346-362). In the presence of ng/ml levels of dsRNA, ATP and divalent cations, the enzyme undergoes autophosphorylation which converts it from a latent to an active protein kinase (Hovanessian, 1989, J. Interferon Res. 9: 641-647; Lebleu, et al., 1976). The autophosphorylation and activation of PKR is prevented by high concentrations of dsRNA (Farrel, et al., 1977; Hunter, et al., 1975, J. Biol. Chem. 250: 409-417). Upon activation, the kinase catalyzes the phosphorylation of the a-subunit (38 kDa) of the eukaryotic initiation factor 2 (eIF-2.alpha.) (Lebleu, et al., 1976; Farrel, et al., 1977; Levin, et al. 1978; Samuel, 1979; Petryshyn, et al., 1983). The phosphorylation of eIF-2.alpha. prevents the ability of eIF-2 to exchange GDP for GTP which is catalyzed by eIF-2.beta. (Matts, et al., 1984, J. Biol. Chem. 259: 6708-6711; Rowlands, et al., 1988, J. Biol. Chem. 263: 5526-5533). This cascade of reactions results in the inhibition of protein synthesis (London, et al., 1987, The Enzymes Vol. 18; Hershey, 1989, J. Biol. Chem. 264: 20823-20826).
Duplex RNA molecules such as reovirus dsRNA and poly(I).cndot.poly(C) (PIC) are well established activators of PKR (Hovanessian, 1989), but the details of the activation process are not fully understood (Galabru, et al., 1989, Eur. J. Biochem. 178: 581-589; Kostura, et al., 1989, Mol. Cell. Biol. 9: 1576-1586; Kitajewski, et al., 1986, Cell 45: 195-200). Moreover, several single stranded (ss) viral RNAs including adenovirus VA1 RNA (Schneider, et al., 1987, Ann. Rev. Biochem. 56: 317-332; Furtado, et al., 1989, J. Virol. 63: 3423-3434; Ghadge, et al., 1994, J. Virol. 68: 4137-4151), Epstein Barr Virus EBER-1 RNA (Clarke, et al., 1991, Nucleic Acids Res. 19: 243-248) and HIV-1 mRNA (Edery, et al., 1989, Cell 56: 303-312; Sen Gupta, et al., 1989, Nucl. Acids Res. 17: 969-978; Roy, et al., 1991, J. Viol. 65: 632-640; Judware, et al., 1993, J. Interferon Res. 13: 153-160) have been demonstrated to contain secondary structures which interact and modulate the activity of PKR. Relatively little is known about the mechanism by which structural elements within these RNAs interact with the kinase but studies have indicated that the amino terminal portion of the protein is important for RNA binding (Feng, et al., 1992, Proc. Natl. Acad. Sci. 89 5447-5451; Manche, et al., 1992, Mol. Cell Biol. 12: 5238-5248; Petryshyn, et al., 1994, Progress in Molecular and Subcellular Biology Vol. 14; Chong, et al., 1992). of significance are the findings that eIF-2.alpha. undergoes phosphorylation in response to addition of cytoplasmic mRNA (Baum, et al., 1983, Biochem. Biophys. Res. Commun 114: 41-49) or polysomal RNA (Pratt, et al., 1988, Nucl. Acids Res. 16: 3497-3510) prepared from uninfected cells and that this phosphorylation is prevented by high concentrations of poly(I).cndot.poly(C) (Baum, et al., 1983; Pratt, et al., 1988). In addition, altered levels of eIF-2 phosphorylation and PKR activity have been reported in some cells subjected to heat-stress conditions (Dubois, et al., 1991, J. Biol. Chem. 266: 9707-9711). Other studies have indicated that the mRNA for PKR itself is capable of facilitating the phosphorylation of PKR (Thomis, et al., 1993, J. Virol. 67: 7695-7700). These observations suggest that some cellular RNAs may regulate the activity of PKR and raise the possibility that the enzyme has a regulatory role in uninfected cells (Petryshyn, et al., 1994). This is supported by the finding of an accumulation of dsRNA capable of activating PKR in embryonal carcinoma cells that have been induced to differentiate but not in uninduced cells (Belkumeur, et al., 1993, Mol. Cell. Biol. 13: 2846-2857). To date, however, the extent and nature of the cellular RNA(s) that mediate the activation of PKR in uninfected cells remain to be identified.
The role of IFN and PKR in the regulation of growth and differentiation of mouse 3T3-F442A cells has recently been investigated. 3T3-F442A cells spontaneously produce and secrete IFN and exhibit a pattern of PKR phosphorylation which is related to specific stages of growth (Petryshyn, et al., 1984). PKR is phosphorylated both in vivo and in vitro in the absence of viral infection or added dsRNA (Petryshyn, et al., 1988). The phosphorylation of PKR is concomitant with increased phosphorylation of eIF-2.alpha., diminished eukaryotic initiation factor 2-.beta. (eIF-2.beta.) activity and a marked reduction in protein synthesis (Petryshyn, et al., 1996, Arch. Biochem. Biophys. 328: 290-297).
A subset of poly(A).sup.+ --rich cytoplasmic RNA that is responsible for activation of PKR in 3T3-F442A cells ("Regulatory RNA" or "R-RNA") has recently been isolated (Li, et al., 1991, Eur. J. Biochem. 195: 41-48) although the nature and exact number of RNAs comprising the R-RNA activity was not determined.
The present invention provides a partial cDNA of about 850 base pairs corresponding to a single and specific cellular R-RNA which, when transcribed in vitro, gives rise to an RNA transcript which retains its property to activate PKR. In addition, the present invention provides a 226-252 nucleotide fragment of the partial cDNA which corresponds to that portion of R-RNA necessary for PKR activation ("the activation sequence"). An antisense molecule to the 226-252 nt cDNA fragment as well as a number of smaller antisense oligonucleotides all of which bind to the sense strand of the R-RNA and thus prevent activation of PKR are also provided. The present invention further provides methods of stimulating the proliferation of hematopoietic cells and fibroblast cells in culture.